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Forchlorfenuron detection based on its inhibitory effect towards catalase immobilized on boron nitride substrate
Biosensors and Bioelectronics 63 (2015) 294–300
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Forchlorfenuron detection based on its inhibitory effect towards catalase immobilized on boron nitride substrate Qin Xu a, Lijuan Cai a, Huijie Zhao b, Jiaqian Tang a, Yuanyuan Shen a, Xiaoya Hu a,n, Haibo Zeng b,n a
College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China Institute of Optoelectronics & Nanomaterials, College of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094, China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 22 April 2014 Received in revised form 12 July 2014 Accepted 22 July 2014 Available online 29 July 2014
An enzymatic procedure based on a catalase biosensor for the detection of forchlorfenuron (CPPU) has been reported in this work. Catalase was immobilized on boron nitride (BN) sheets dispersed in chitosan by adsorption. The immobilized catalase exhibited direct electron transfer character and excellent electrocatalytic activity towards H2O2 reduction. After introducing CPPU into the H2O2 containing phosphate buffer solution, the catalase-catalyzed H2O2 reduction current decreased. By measuring the current decrease, CPPU can be determined in the range of 0.5–10.0 mM with the detection limit of 0.07 μM. The non-competitive inhibition behavior of CPPU towards catalase was verified by the Lineweaver–Burk plots. Long stability character has been ascribed to this biosensor. Possible use of this biosensor in flow systems is illustrated. The proposed biosensor has been successfully applied to CPPU determination in fruits samples with satisfactory results. & Elsevier B.V. All rights reserved.
Keywords: Forchlorfenuron Inhibition Catalase Boron nitride
1. Introduction Forchlorfenuron, also named as 1-(2-chloropyridin-4-yl)-3phenylurea (CPPU), is a kind of plant growth hormone that is currently utilized to accelerate cell division, expand cell volume and promote organ formation and protein synthesis (Nishijima and Shima, 2006). CPPU has been widely used due to the increasing demand for high-quality fruits in the international market, so it is possible that the residues of this agrochemical could eventually reach the consumers. However, the US Code of Federal Regulations defined that the maximum residue limit (MRL) of CPPU should not exceed 200.0 nM in all food commodities (US Electronic Code of Federal Regulations; Title 40). Long-term exposure to CPPU could cause the body protein metabolism disorder, mild emphysema and thin (Hu et al., 2008). In this respect, CPPU residues in food should be monitored and controlled. Thus, it is necessary to develop analytical tools to identify CPPU-positive samples. Different analytical methods have been employed to evaluate CPPU concentration such as chromatography combined with UV (Hu and Li, 2006) or MS detectors (Shi et al., 2012) and n
Corresponding authors. E-mail addresses: [email protected] (X. Hu), [email protected] (H. Zeng). http://dx.doi.org/10.1016/j.bios.2014.07.055 0956-5663/& Elsevier B.V. All rights reserved.
immunoassay (Suárez-Pantaleon et al., 2012). Suárez-Pantaleon et al. (2012) have summarized these up-to-date published strategies. Despite the sensitive and selective of these traditionally employed analytical technologies, they have some drawbacks such as the cost and sophistication of the instrumentation, highly qualified personnel requirement. Alternatively, biosensors represent a rapid, cost effective and simple methodology because they possess advantages such as screening of various contaminants in environmental matrices, minimizing the sample pretreatment, reducing cost and time of analysis, and displaying sufficient sensitivity and selectivity. Electrochemical biosensors based on the inhibition of different enzymes have been reported (Ge et al., 2013). Catalase is an ideal enzyme for inhibition studies because it presents in all aerobic and many anaerobic organisms. As a biological catalyst, catalase has remarkable advantages such as high efficient and selective catalytic performance under mild conditions, low cost, good stability and high specific activity. However most of the catalase based biosensors cited in the literature are developed for the determination of their substrates (Chen et al., 2001; Huang et al., 2011; Zhou et al., 2008). Only a few reports are devoted to the determination of specific inhibitors (Singh et al., 2009). Boron nitride (BN) is isoelectronic to carbon. It has a stable hexagonal structure analogous to that of graphite. The large thermal conductivity, superior temperature stability and acid–base resistance properties make BN invaluable for high-performance
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Scheme 1. Illustration of the construction of catalase/BN/chitosan biosensor and its operation for CPPU detection.
nanocomposites materials. BN is also found to be nontoxic to health and environment due to its chemical inertness and structural stability. Therefore, BN is more suitable for biosensor construction (Chen et al., 2009). However, the potential applications of BNs for biosensors are hampered for their poor dispersion in many aqueous and organic solvents because they hold highly hydrophobic sidewalls, and would aggregate tightly via van der Waals forces (Xie et al., 2005). A few efforts have been addressed to disperse BNs in aqueous phases, for example the noncovalent surface treatment of BN by surfactant or polymers (Zhi et al., 2005). Chitosan is an interesting polymer which has been reported to improve the dispersion of multiwall carbon nanotubes (CNTs) due the hydrophobic interaction of the hydrocarbon chains of chitosan and the π bonds of the nanotube (Iamsamai et al., 2010). Chitosan also has good biocompatibility and excellent film forming ability which made it suitable for the construction of biosensors (Liu et al., 2005). In this work, chitosan was used to increase the solubility of BN in aqueous solution. Then catalase was immobilized onto the well-dispersed BN, and the immobilized catalase was modified on an electrode to construct a biosensor for CPPU determination. Scheme 1 illustrates the construction and operation process of the biosensor. Catalase immobilized on BN exhibited excellent bioelectrocatalytic activity towards the reduction of H2O2. However, the activity of catalase would be inhibited proportionally by CPPU which would cause the decrease of the H2O2 reduction current. The percentage of catalase inhibition was used for the quantitative detection of CPPU. Detailed characterization, optimization and application studies of this biosensor have been discussed.
Working solutions of CPPU were obtained by serial dilution of the stock solution into 0.1 M phosphate buffer solution. The fruits (kiwifruit, watermelon and grape) and orange juice sample (minute maid pulpy) were bought from local supermarket. BN was supplied by Nanjing University of Science and Technology. All other chemicals not mentioned here were of analytical reagent grade and were used without further purification. Double-distilled water was used throughout the experiment. 0.1 M phosphate buffer solution was prepared by mixing 0.1 M Na2HPO4 and 0.1 M NaH2PO4 with a volume ratio of 1:1 and then adjusted to the desired pH by NaOH or H3PO4. The morphologies of BN, BN/chitosan, catalase/BN and catalase/ BN/chitosan were investigated by using a Hitachi S-4800 Field Emission Scanning Electron Microscope (Hitachi Co., Japan). The cyclic voltammetric (CV) and amperometric measurements were performed on a CHI 840 electrochemical analyzer (Shanghai Chenhua, China), using a conventional three-electrode system with a platinum foil as the auxiliary electrode and a saturated calomel electrode (SCE) as the reference electrode. Working electrodes were bare or modified glassy carbon electrodes (GCE) (3 mm in diameter, Shanghai Chenhua, China). Before each experiment, the GCE was polished with 0.3 and 0.05 mm alumina slurries, rinsed with doubly distilled water, and then purified sequentially by HNO3 (1:1, v/v), ethanol (1:1, v/v) and distilled water. A 25 mL glass electrochemical cell was used for batch electrochemical measurements. The flow injection apparatus consisted of a peristaltic pump (Ismatec ISM 828), a homemade rotatory injection valve and an electrochemical flow-cell (Chenhua, Shanghai). 2.2. Preparation of BN and different electrodes
2. Experimental 2.1. Reagents and apparatus Catalase from bovine liver (E.C. 1.11.1.6) was purchased from Sigma and was used without further purification. Thiourea, ammonia borane, hydrogen peroxide (H2O2) (33%) and chitosan were purchased from Shanghai Chemical Reagent Co. (Shanghai, China). CPPU was obtained from Aladdin (Shanghai, China). The standard CPPU stock solution (0.100 M) was prepared in anhydrous ethanol.
The detailed process for the preparation of BN sheet was as follows. In brief, the vesicant of thiourea and the precursors of ammonia borane (AB) with a mass ratio of 0.4:1 were heated at a rate of 8 °C min 1 from room temperature to 1200 °C and kept there for 3 h under a flow of N2 (100 sccm), then natural cooling to room temperature, hence we can acquire the high quality BN product without complex feedstock pretreatment and follow-up treatment. In this work, chitosan was used to help the stable dispersion of BN in aqueous solution due to its good biocompatibility and
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excellent film forming ability (Iamsamai et al., 2010; Liu et al., 2005). Chitosan solution (0.5%) (w/v) was prepared by dissolving 0.5 g chitosan powder in 100.0 mL of 1.0% acetic acid solution. Then 1.0 mg of BN was added into 1.0 mL of 0.5% chitosan solution to form a homogeneous dispersion with ultrasonication. 50.0 μL of 8.0 mg/ml of catalase solution was placed into 50.0 μL of 1.0 mg/mL of BN suspension, and the mixture was shaken strongly for about 30 min at room temperature for the adsorption of catalase. After the adsorption process, 5.0 μL of the above obtained mixture was spread evenly onto the surface of the pretreated glassy carbon electrode by a syringe. Then the electrode was left to dry at 4 °C. The resulting electrode was marked as catalase/BN/ chitosan/GCE. When not in use, the modified electrode was stored at 4 °C. For comparison, BN dispersed in water was also used for the immobilization of catalase by the same procedure, and the electrode was labeled as catalase/BN/GCE. 2.3. Sample preparation An amount of 1.0 kg of each fruit sample was peeled before being chopped, ground and homogenized using a high-speed blender. The samples were placed in closed polyethylene tubes at 4 °C before analysis. Then 10.0 g portion of fruit samples were placed in a centrifuge tube and 10.0 mL of chloroform was added as well. After that, the tube was vigorously shaken for 2 min. The extract was obtained by centrifugation for 10 min. Finally, an extract containing the equivalent 1.0 g of sample/mL in chloroform was obtained. Then, this extract was evaporated to dryness under a flow of nitrogen at room temperature. The dry residues were dissolved in 1.0 mL of ethanol. The obtained solution was centrifuged at 4000 rpm for 2 min. Samples used for recovery studies were previously tested by the HPLC method and proved to be free from CPPU. The above described method was used for preparation of blank fruit samples and spiked with a known quantity of CPPU for recovery studies.
2.4. Measurements procedures Cyclic voltammetric (CV) measurements were performed with a CHI 840 electrochemical workstation in a 25.0 mL glass cell. 10.0 mL of 0.1 M pH 7.0 phosphate buffered solution was used as an electrolyte solution. Prior to each experiment, buffer solutions were deaerated by continuous bubbling of N2 gas for at least 30 min. During the experiments, N2 was continuously passed over the solution in order to maintain inert atmosphere. For inhibition studies, flow injection measurements with amperometric detection were carried out. Different concentrations of CPPU were injected into the stream consisting of 1.0 mM of H2O2 and 0.1 M phosphate buffered solution (pH 7.0) with a flow rate of 3.0 mL min 1. Subsequently, the sample zone flowed through the detection unit with an injection volume of 50.0 mL, and the signal was monitored by a CHI 840 electrochemical analyzer at an applied potential of 0.35 V versus SCE. The current generated as a result of the reduction of H2O2 is correlated to CPPU concentrations. The percentage of catalase inhibition (%ICAT) by different concentrations of CPPU was calculated using Eq. (1)
%ICAT (%) =
(Ii ℬ IF ) ¬ 100 Ii
(1)
where Ii and IF are the currents of the biosensor obtained in the absence and presence of CPPU, respectively. After each inhibition experiment, the working electrode was soaked into phosphate buffed solution (pH 7.0) for 5 min to reactivate it. High-performance liquid chromatography (HPLC) determinations were conducted using a 1.0 mL min 1 methanol/water flow with a gradient from 40% to 90% (v/v) methanol in 15 min, and then 90% (v/v) methanol was run during 5 min. A 20.00 μL injection volume was employed. Measurement wavelengths were 256 nm for CPPU.
Fig. 1. (A) SEM image of BN dispersed in water solution, inset was the photograph of BN–water solution; (B) SEM image of BN dispersed in 0.5% (w/v) chitosan solution, inset was the photograph of BN–chitosan solution; (C) SEM image of catalase immobilized in BN which was dispersed in water (catalase/BN) and (D) SEM image of catalase immobilized in BN which was dispersed in chitosan solution (catalase/BN/chitosan).
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3. Results and discussion 3.1. Characterization of BN, BN/chitosan, catalase/BN and catalase/ BN/chitosan In this work, BN dispersed in chitosan solution was used to adsorb catalase. Integrating the merits of BN and chitosan, BN dispersed in chitosan was more biocompatible and it offered a favorable microenvironment for the immobilization of catalase. Fig. 1A shows the sheet structure of the synthesized BN. These BN sheets aggregated together in aqueous solution with poor dispersion (inset of Fig. 1A). Fig. 1B shows that BN dispersed well in chitosan matrix. A stable milky solution was formed when 0.5% chitosan solution was used for the dispersion of the synthesized BN sheets (inset of Fig. 1B). The well-dispersed BN/chitosan can form a stable solution for a week without obvious clustering or precipitation. Chitosan is a kind of natural polyaminosaccharide. The hydrophilic/hydrophobic nature of chitosan makes it suitable to improve the solubility of BN in aqueous solution (Kisku and Swain, 2012). Its linear polyglucosamine chains have a strong tendency to adsorb on BN sheet via van der Waals forces and hydropholic interactions, and the hydrophilic amino and hydroxyl ligand helps to disperse the functionalized BN in water solution. The morphology of catalase/BN is also observed by using SEM. As shown in Fig. 1C, catalase biomolecules are excellently dispersed on BN without any aggregation, suggesting that BN is a suitable matrix for the immobilization of catalase. Fig. 1D shows that the catalase/BN/chitosan film is continuous and more uniform, which is advantageous to retain the bioactivity of proteins. UV–vis spectroscopy is a useful tool to study the possible conformational change of catalase in BN sheets. Fig. S1 shows the UV–vis absorption spectra of BN (curve a), catalase/BN (curve b) and catalase (curve c). In the case of catalase, the absorption band at about 270 nm originates from the aromatic amino acids, i.e., tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe). The changes observed in this region give evidence of the rearrangement in the globule structure of catalase (Bartoszek and Su, 2006). The position and shape of the absorption bands for catalase/BN are almost the same as those of pure catalase, suggesting that the catalase immobilized onto BN indeed maintains its native structure. 3.2. Electrochemical behavior of catalase/BN/chitosan biosensor
Fig. 2. (A) CVs of the bare GCE (curve a), BN/chitosan/GCE (curve b), catalase/ chitosan/GCE (curve c), catalase/BN/GCE and catalase/BN/chitosan/GCE (curve d) in N2-saturated phosphate buffered solution (pH 7.0, 0.1 M) at the scan rate of 100 mV/s; (B) CVs of the catalase/BN/chitosan/GCE in N2-saturated phosphate buffered solution (pH 7.0, 0.1 M) in the absence of H2O2 (a); in the presence of 100.0 μM H2O2 (b); the same as b with 5.0 μM (c) and 10.0 μM (d) of CPPU. Scan rate: 100 mV/s.
of the immobilized catalase. The catalase/BN/GCE was not stable. The modified film would deplete away from the electrode after 20 cycles. Thus chitosan was used to help the dispersion of BN in aqueous solution and to improve the stability of catalase/BN on the electrode because of its good film forming ability. It is well known that catalase-modified electrodes exhibit the electrocatalytic activities for the reduction of H2O2. Fig. 2B shows the CV response of the catalase/BN/chitosan/GCE in 0.1 M pH 7.0 phosphate buffered solution in the absence (curve a) and presence of 100.0 μM H2O2 (curve b). As compared with curve a, curve b shows larger peak current, indicating that catalase exhibits excellent bioelectrocatalytic activity for the destruction of H2O2. The general mechanism of catalase for the decomposition of H2O2 is as follows (Varma and Mattiasson, 2005):
H2 O2 + catℬFe(III) ћ H2 O+ catℬFe(IV)=O
(Reaction 1)
compound-I Fig. 2A shows cyclic voltammograms (CVs) of the bare-GCE (curve a), BN/chitosan/GCE (curve b), catalase/chitosan/GCE (curve c), catalase/BN/GCE (curve d) and catalase/BN/chitosan/GCE (curve e) in deoxygenized 0.1 M pH 7.0 phosphate buffered solution at the scan rate of 100 mV/s. For bare GCE (curve a), BN/chitosan/GCE (curve b) and catalase/chitosan/GCE (curve c), no redox peak is observed, indicating that all of them cannot undergo the redox reaction in the investigated potential range. For catalase/BN/GCE (curve d) and catalase/BN/chitosan/GCE (curve e), a pair of welldefined nearly reversible redox peaks with a formal potential of about 0.320 V versus SCE was observed. The observed formal potential for the present catalase/BN/GCE or catalase/BN/chitosan/ GCE is almost the same to the reported values for other catalasemodified electrodes (Shamsipur et al., 2012; Zhou et al., 2008). The peaks can be attributed to the redox reaction of the Cat-Fe(III)/CatFe(IV) redox couple at the active site of the immobilized catalase (Jones and Suggett, 1968; Nam, 2007; Varma and Mattiasson, 2005). This suggests that BN between the catalase and the GCE has assisted the direct electron transfer from the active site of catalase to the electrode. However, the response of catalase on BN in the absence of chitosan was smaller because of the poor dispersion of BN in aqueous solution thus caused the less amount
H2 O2 + CatℬFe(IV) = Oћ H2 O+ O2 + catℬFe(III)
(Reaction 2)
Reaction 1 is the rate-determining step. Once catalase compound-I forms (Reaction 1), it rapidly reacts with a second molecule of H2O2 to generate O2 and a water molecule (Reaction 2) thus causes the increase of the currents. Primary purpose of this study is to develop a catalase based biosensor for CPPU detection. Thus, the inhibitory effects of CPPU on the catalase catalyzed H2O2 reduction were investigated. Curves c and d in Fig. 2B show that the reduction peak current of H2O2 on catalase/BN/chitosan biosensor decreased with the increase of the concentration of the added CPPU. Thus, it is reasonable to say that the decrease in the catalytic cathodic current of the catalase/BN/chitosan biosensor in the presence of CPPU would be originated from the inactivation of the catalytic activity of catalase for H2O2 reduction. Namely, CPPU binding to catalase inhibits the reaction of H2O2 with catalase (Varma and Mattiasson, 2005), resulting in the change of the CV responses. The decrease in the peak current is directly proportional to the concentration of the inhibitor in the test solution because the percentage of inhibited enzyme that results after exposure to the
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Fig. 3. Double-reciprocal Lineweaver–Burk plot CPPU inhibition. (a) 0.0 M CPPU; (b) 2.0 μM CPPU; (c) 4.0 μM CPPU. (B) Schematic diagram of the interaction between CPPU and catalase.
inhibitor is quantitatively related to the inhibitor concentration (Amine et al., 2006). 3.3. Investigation on the type of inhibition Enzyme inhibition is a reaction between a molecule and an enzyme that blocks the action of the enzyme, either temporarily or permanently. There are several types of inhibition involving competitive, non-competitive and uncompetitive or mixed inhibition. The double-reciprocal (also known as the Lineweaver–Burk) plot was used to study the inhibition type when the concentration of H2O2 was varied at several fixed concentrations of CPPU. Fig. 3A shows a series of lines converging on the same point on the X while the Y-intercept of the plots increases with the increase of CPPU concentration. For Lineweaver–Burk plot, the slope of the resulting line is KM/Vmax, the y-intercept is 1/Vmax, and the xintercept is 1/KM. The inhibition of catalase by CPPU causes a decrease in Vmax value while KM is unaffected. The hallmarks of noncompetitive inhibition are an unchanging Michaelis constant (KM) and a decrease of the maximum velocity (Vmax) when the inhibitor is present (Fersht, 1985). So CPPU acts as a noncompetitive inhibitor by binding to catalase. Fig. 3B was used to illustrate the inhibition mechanism. In this figure, E represents the catalase enzyme; S represents H2O2 and I represents CPPU. CPPU binds to catalase at a location other than the active site. 3.4. Optimization of the variables concerning the catalase/BN/ chitosan biosensor under flow-injection conditions The experimental variables, which can affect the detection of CPPU, including the catalase to BN ratio, pH of the supporting electrolyte; applied potential and the concentration of substrate were investigated. To obtain good sensitivity of the sensor, catalase to BN ratio was optimized. Fig. S2A shows the relationship between the catalase to BN ratio and the inhibition degree of catalase by 5.0 mM CPPU. The result shows that the maximum inhibition degree was attained when the catalase to BN ratio was 8:1. It means that 1.0 mg/mL BN was the optimized concentration for the immobilization of catalase when 8.0 mg/mL catalase was used. Thus, this optimized ratio was used in all the following experiments.
Fig. S2B shows the relationship between pH and the inhibition degree of catalase by 5.0 mM CPPU. It is found the inhibition degree of catalase/BN/chitosan biosensor rises as pH increases before pH 7.0; when pH value is higher than 7.0, the inhibition degree drops as pH increases because the enzyme loses its activity. This is similar to that reported for catalase immobilized on amidoxime polyacrylonitrile nanofibrous membranes (Feng et al., 2013) and amine-functionalized graphene/gold nanoparticles composite (Huang et al., 2011). So pH 7.0 is chosen as the optimum pH of this inhibition study. The applied potential has an important influence on the sensitivity and selectivity of the biosensor. Fig. S2C shows the relationship between the applied potential and the inhibition degree of catalase by 5.0 mM CPPU. When the applied potential is decreased from 0.25 to 0.35 V, the inhibition degree increases because of the increasing driving force for the fast reduction of H2O2. When the applied potential is more negative than 0.35 V, the inhibition degree decreases with decreasing the applied potential. It can be due to a potential dependence of the activity of the immobilized catalase. In more negative potential values, catalase could be inactivated due to the formation of a high formal oxidation state of enzyme, commonly known as compound-III (Fe III) (Csöregi et al., 1993), thus caused the decrease of its catalytic effect towards H2O2. The inhibition degree also decreased. So, 0.35 V was used as the detection potential for further studies. For an inhibitor biosensor, the effect of the substrate concentration has to be adjusted carefully to obtain better results. Table S1 shows the effect of H2O2 concentration on the inhibitory influence of CPPU to catalase/BN/chitosan biosensor. The detection limit of CPPU increased with increasing the concentration of H2O2. The substrate will compete with inhibitor when the substrate concentration is high, so the increase of substrate concentration will lead to the decrease of inhibition of inhibitor on the enzyme. Otherwise, when the substrate concentration is low, the electrochemical response generated by the enzyme electrode is so weak that the determination of CPPU is hardly feasible. Therefore, we chose 1.0 mM H2O2 as the suitable substrate concentration for inhibition study. 3.5. Performance characteristics of the catalase/BN/chitosan biosensor When the constructed catalase/BN/chitosan biosensor was used at the above mentioned conditions in a flow system, the produced current would change with different concentrations of CPPU. Fig. 4 illustrates the effect of CPPU on the catalase-catalyzed H2O2 reduction peak currents. CPPU displayed increasing inhibition on the activity of catalase with increasing concentration. The inhibition curve tented to stable when the concentration of CPPU is large enough. However, the maximum inhibition value of CPPU was not 100%, which indicated that the binding sites between CPPU and enzymes could reach saturation and equilibrium. The inhibition degree (I%) of the catalase/BN/chitosan biosensor has a linear relationship with the concentration of CPPU from 0.50 to 10.0 μM. The regression equation was ΔI (μA)¼0.0083 þ 0.049C (μM) with a correlation coefficient of 0.999. The limit of the detection (LOD) for CPPU by the present catalase/BN/chitosan flow-biosensor was found to be 0.07 μM base on S/N ¼3. Table S2 listed the analytical performance of different methods for CPPU detection. This method is sensitive than the HPLC–UV method (Kobayashi et al., 2007), but is less sensitive than LC–ESI-TOFMS (Campillo et al., 2013) and ELISA methods (Suárez-Pantaleón et al., 2013). However, this value was below the approximately CPPU concentration claimed by US Code of Federal Regulations. Therefore, the proposed method is suitable for testing CPPU in real samples.
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addition method. HPLC with UV detector was chosen as a comparison method to evaluate the validity of the proposed procedure. The results obtained by the catalase/BN/chitosan biosensor and the HPLC method were summarized in Table 1. There were agreed well with each other, implying that the developed method was capable of practical applications.
4. Conclusions
Fig. 4. Flow-injection amperometric curves of catalase/BN/chitosan biosensor for different concentrations of CPPU. Inset was the calibration curves between CPPU and inhibition efficiency. The experiment was performed in 0.1 M phosphate buffered solution (pH 7.0) containing 1.0 mM H2O2 at 0.35 V; the data represented mean values and standard deviations of five measurements.
The high selectivity of the established method toward CPPU detection is important whether juice, fruit or other foods are being investigated. A systematic study of sample solutions containing a fixed amount of CPPU (1.0 mM) spiked with excess amount of some common interferences that are coexsisted with CPPU in fruits and juice samples was made to know the effect of interferences on the efficiency and selectivity of the proposed analytical method to CPPU detection. No influence on the detection of 1.0 mM CPPU was found after the addition of 1000-fold concentrations of glucose, sucrose, glycine, citric acid, Na þ and Ca2 þ . Thus the proposed method provides a possible way to monitor CPPU. The repeatability of the measurement was calculated for thirty independent runs of 1.0 μM CPPU. The relative standard deviation was 7.89%. The reproducibility of the biosensor was evaluated by the response variation of 10 biosensors, prepared on different days. The relative standard deviation was 9.73% (n ¼10), indicating good reproducibility of the sensor. The stability of the catalase/BN/ chitosan flow-biosensor was investigated by storing it at 4 °C in refrigerator and checked its response to 1.0 μM CPPU everyday. The biosensor maintained 91.3% of its initial activity after two months. Thus the presence of BN is very efficient for retaining the enzyme activity of catalase 3.6. Real sample analysis To illustrate the feasibility of the developed method for routine analysis, the method was applied to determine CPPU in different fruits and juices samples. Because the existing fruits and juices samples in the market are free of CPPU, they were fortified at different concentrations of CPPU, ranging from 1.0 to 10.0 μM. Each sample was determined in triplicate using the standard Table 1 Measurement results of CPPU in fruits and juice samples (n¼ 3). Analyte
Orange Juice Kiwifruit Watermelon Grape
Spiked (μM)
5.00 4.00 1.00 8.00
Found (mM) Catalase/BN/ chitosan biosensor
HPLC
Recovery by catalase/ BN/chitosan biosensor (%)
4.76 4.19 0.94 7.46
4.77 3.93 0.92 7.87
95.20 104.7 94.00 93.25
This work showed that the catalytic activity of catalase immobilized on BN to the reduction of H2O2 can be inhibited by CPPU. A simple and effective biosensor for the detection of CPPU has been constructed based on this inhibition effect. This biosensor has the advantages such as suitability for detecting in small volumes, fast response, low detection limit and ease of regeneration. Moreover, the presented sensor has demonstrated good sensitivity, reusability and reproducibility. These characters demonstrated the possibility of applying the biosensor, together with flow injection analysis, for the rapid determination of CPPU at low cost.
Acknowledgment The authors gratefully acknowledge the National Basic Research Program of China (2014CB931700), NSFC (21275124, 21275125 and 61222403), the Foundation of Jiangsu Educational Bureau (12KJB150022), the Qinlan Project of JiangSu Province (Grant no. 11KJB150019), and the project funded by the PAPD.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.055.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Forchlorfenuron detection based on its inhibitory effect towards catalase immobilized on boron nitride substrate Qin Xu a, Lijuan Cai a, Huijie Zhao b, Jiaqian Tang a, Yuanyuan Shen a, Xiaoya Hu a,n, Haibo Zeng b,n a
College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China Institute of Optoelectronics & Nanomaterials, College of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094, China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 22 April 2014 Received in revised form 12 July 2014 Accepted 22 July 2014 Available online 29 July 2014
An enzymatic procedure based on a catalase biosensor for the detection of forchlorfenuron (CPPU) has been reported in this work. Catalase was immobilized on boron nitride (BN) sheets dispersed in chitosan by adsorption. The immobilized catalase exhibited direct electron transfer character and excellent electrocatalytic activity towards H2O2 reduction. After introducing CPPU into the H2O2 containing phosphate buffer solution, the catalase-catalyzed H2O2 reduction current decreased. By measuring the current decrease, CPPU can be determined in the range of 0.5–10.0 mM with the detection limit of 0.07 μM. The non-competitive inhibition behavior of CPPU towards catalase was verified by the Lineweaver–Burk plots. Long stability character has been ascribed to this biosensor. Possible use of this biosensor in flow systems is illustrated. The proposed biosensor has been successfully applied to CPPU determination in fruits samples with satisfactory results. & Elsevier B.V. All rights reserved.
Keywords: Forchlorfenuron Inhibition Catalase Boron nitride
1. Introduction Forchlorfenuron, also named as 1-(2-chloropyridin-4-yl)-3phenylurea (CPPU), is a kind of plant growth hormone that is currently utilized to accelerate cell division, expand cell volume and promote organ formation and protein synthesis (Nishijima and Shima, 2006). CPPU has been widely used due to the increasing demand for high-quality fruits in the international market, so it is possible that the residues of this agrochemical could eventually reach the consumers. However, the US Code of Federal Regulations defined that the maximum residue limit (MRL) of CPPU should not exceed 200.0 nM in all food commodities (US Electronic Code of Federal Regulations; Title 40). Long-term exposure to CPPU could cause the body protein metabolism disorder, mild emphysema and thin (Hu et al., 2008). In this respect, CPPU residues in food should be monitored and controlled. Thus, it is necessary to develop analytical tools to identify CPPU-positive samples. Different analytical methods have been employed to evaluate CPPU concentration such as chromatography combined with UV (Hu and Li, 2006) or MS detectors (Shi et al., 2012) and n
Corresponding authors. E-mail addresses: [email protected] (X. Hu), [email protected] (H. Zeng). http://dx.doi.org/10.1016/j.bios.2014.07.055 0956-5663/& Elsevier B.V. All rights reserved.
immunoassay (Suárez-Pantaleon et al., 2012). Suárez-Pantaleon et al. (2012) have summarized these up-to-date published strategies. Despite the sensitive and selective of these traditionally employed analytical technologies, they have some drawbacks such as the cost and sophistication of the instrumentation, highly qualified personnel requirement. Alternatively, biosensors represent a rapid, cost effective and simple methodology because they possess advantages such as screening of various contaminants in environmental matrices, minimizing the sample pretreatment, reducing cost and time of analysis, and displaying sufficient sensitivity and selectivity. Electrochemical biosensors based on the inhibition of different enzymes have been reported (Ge et al., 2013). Catalase is an ideal enzyme for inhibition studies because it presents in all aerobic and many anaerobic organisms. As a biological catalyst, catalase has remarkable advantages such as high efficient and selective catalytic performance under mild conditions, low cost, good stability and high specific activity. However most of the catalase based biosensors cited in the literature are developed for the determination of their substrates (Chen et al., 2001; Huang et al., 2011; Zhou et al., 2008). Only a few reports are devoted to the determination of specific inhibitors (Singh et al., 2009). Boron nitride (BN) is isoelectronic to carbon. It has a stable hexagonal structure analogous to that of graphite. The large thermal conductivity, superior temperature stability and acid–base resistance properties make BN invaluable for high-performance
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Scheme 1. Illustration of the construction of catalase/BN/chitosan biosensor and its operation for CPPU detection.
nanocomposites materials. BN is also found to be nontoxic to health and environment due to its chemical inertness and structural stability. Therefore, BN is more suitable for biosensor construction (Chen et al., 2009). However, the potential applications of BNs for biosensors are hampered for their poor dispersion in many aqueous and organic solvents because they hold highly hydrophobic sidewalls, and would aggregate tightly via van der Waals forces (Xie et al., 2005). A few efforts have been addressed to disperse BNs in aqueous phases, for example the noncovalent surface treatment of BN by surfactant or polymers (Zhi et al., 2005). Chitosan is an interesting polymer which has been reported to improve the dispersion of multiwall carbon nanotubes (CNTs) due the hydrophobic interaction of the hydrocarbon chains of chitosan and the π bonds of the nanotube (Iamsamai et al., 2010). Chitosan also has good biocompatibility and excellent film forming ability which made it suitable for the construction of biosensors (Liu et al., 2005). In this work, chitosan was used to increase the solubility of BN in aqueous solution. Then catalase was immobilized onto the well-dispersed BN, and the immobilized catalase was modified on an electrode to construct a biosensor for CPPU determination. Scheme 1 illustrates the construction and operation process of the biosensor. Catalase immobilized on BN exhibited excellent bioelectrocatalytic activity towards the reduction of H2O2. However, the activity of catalase would be inhibited proportionally by CPPU which would cause the decrease of the H2O2 reduction current. The percentage of catalase inhibition was used for the quantitative detection of CPPU. Detailed characterization, optimization and application studies of this biosensor have been discussed.
Working solutions of CPPU were obtained by serial dilution of the stock solution into 0.1 M phosphate buffer solution. The fruits (kiwifruit, watermelon and grape) and orange juice sample (minute maid pulpy) were bought from local supermarket. BN was supplied by Nanjing University of Science and Technology. All other chemicals not mentioned here were of analytical reagent grade and were used without further purification. Double-distilled water was used throughout the experiment. 0.1 M phosphate buffer solution was prepared by mixing 0.1 M Na2HPO4 and 0.1 M NaH2PO4 with a volume ratio of 1:1 and then adjusted to the desired pH by NaOH or H3PO4. The morphologies of BN, BN/chitosan, catalase/BN and catalase/ BN/chitosan were investigated by using a Hitachi S-4800 Field Emission Scanning Electron Microscope (Hitachi Co., Japan). The cyclic voltammetric (CV) and amperometric measurements were performed on a CHI 840 electrochemical analyzer (Shanghai Chenhua, China), using a conventional three-electrode system with a platinum foil as the auxiliary electrode and a saturated calomel electrode (SCE) as the reference electrode. Working electrodes were bare or modified glassy carbon electrodes (GCE) (3 mm in diameter, Shanghai Chenhua, China). Before each experiment, the GCE was polished with 0.3 and 0.05 mm alumina slurries, rinsed with doubly distilled water, and then purified sequentially by HNO3 (1:1, v/v), ethanol (1:1, v/v) and distilled water. A 25 mL glass electrochemical cell was used for batch electrochemical measurements. The flow injection apparatus consisted of a peristaltic pump (Ismatec ISM 828), a homemade rotatory injection valve and an electrochemical flow-cell (Chenhua, Shanghai). 2.2. Preparation of BN and different electrodes
2. Experimental 2.1. Reagents and apparatus Catalase from bovine liver (E.C. 1.11.1.6) was purchased from Sigma and was used without further purification. Thiourea, ammonia borane, hydrogen peroxide (H2O2) (33%) and chitosan were purchased from Shanghai Chemical Reagent Co. (Shanghai, China). CPPU was obtained from Aladdin (Shanghai, China). The standard CPPU stock solution (0.100 M) was prepared in anhydrous ethanol.
The detailed process for the preparation of BN sheet was as follows. In brief, the vesicant of thiourea and the precursors of ammonia borane (AB) with a mass ratio of 0.4:1 were heated at a rate of 8 °C min 1 from room temperature to 1200 °C and kept there for 3 h under a flow of N2 (100 sccm), then natural cooling to room temperature, hence we can acquire the high quality BN product without complex feedstock pretreatment and follow-up treatment. In this work, chitosan was used to help the stable dispersion of BN in aqueous solution due to its good biocompatibility and
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excellent film forming ability (Iamsamai et al., 2010; Liu et al., 2005). Chitosan solution (0.5%) (w/v) was prepared by dissolving 0.5 g chitosan powder in 100.0 mL of 1.0% acetic acid solution. Then 1.0 mg of BN was added into 1.0 mL of 0.5% chitosan solution to form a homogeneous dispersion with ultrasonication. 50.0 μL of 8.0 mg/ml of catalase solution was placed into 50.0 μL of 1.0 mg/mL of BN suspension, and the mixture was shaken strongly for about 30 min at room temperature for the adsorption of catalase. After the adsorption process, 5.0 μL of the above obtained mixture was spread evenly onto the surface of the pretreated glassy carbon electrode by a syringe. Then the electrode was left to dry at 4 °C. The resulting electrode was marked as catalase/BN/ chitosan/GCE. When not in use, the modified electrode was stored at 4 °C. For comparison, BN dispersed in water was also used for the immobilization of catalase by the same procedure, and the electrode was labeled as catalase/BN/GCE. 2.3. Sample preparation An amount of 1.0 kg of each fruit sample was peeled before being chopped, ground and homogenized using a high-speed blender. The samples were placed in closed polyethylene tubes at 4 °C before analysis. Then 10.0 g portion of fruit samples were placed in a centrifuge tube and 10.0 mL of chloroform was added as well. After that, the tube was vigorously shaken for 2 min. The extract was obtained by centrifugation for 10 min. Finally, an extract containing the equivalent 1.0 g of sample/mL in chloroform was obtained. Then, this extract was evaporated to dryness under a flow of nitrogen at room temperature. The dry residues were dissolved in 1.0 mL of ethanol. The obtained solution was centrifuged at 4000 rpm for 2 min. Samples used for recovery studies were previously tested by the HPLC method and proved to be free from CPPU. The above described method was used for preparation of blank fruit samples and spiked with a known quantity of CPPU for recovery studies.
2.4. Measurements procedures Cyclic voltammetric (CV) measurements were performed with a CHI 840 electrochemical workstation in a 25.0 mL glass cell. 10.0 mL of 0.1 M pH 7.0 phosphate buffered solution was used as an electrolyte solution. Prior to each experiment, buffer solutions were deaerated by continuous bubbling of N2 gas for at least 30 min. During the experiments, N2 was continuously passed over the solution in order to maintain inert atmosphere. For inhibition studies, flow injection measurements with amperometric detection were carried out. Different concentrations of CPPU were injected into the stream consisting of 1.0 mM of H2O2 and 0.1 M phosphate buffered solution (pH 7.0) with a flow rate of 3.0 mL min 1. Subsequently, the sample zone flowed through the detection unit with an injection volume of 50.0 mL, and the signal was monitored by a CHI 840 electrochemical analyzer at an applied potential of 0.35 V versus SCE. The current generated as a result of the reduction of H2O2 is correlated to CPPU concentrations. The percentage of catalase inhibition (%ICAT) by different concentrations of CPPU was calculated using Eq. (1)
%ICAT (%) =
(Ii ℬ IF ) ¬ 100 Ii
(1)
where Ii and IF are the currents of the biosensor obtained in the absence and presence of CPPU, respectively. After each inhibition experiment, the working electrode was soaked into phosphate buffed solution (pH 7.0) for 5 min to reactivate it. High-performance liquid chromatography (HPLC) determinations were conducted using a 1.0 mL min 1 methanol/water flow with a gradient from 40% to 90% (v/v) methanol in 15 min, and then 90% (v/v) methanol was run during 5 min. A 20.00 μL injection volume was employed. Measurement wavelengths were 256 nm for CPPU.
Fig. 1. (A) SEM image of BN dispersed in water solution, inset was the photograph of BN–water solution; (B) SEM image of BN dispersed in 0.5% (w/v) chitosan solution, inset was the photograph of BN–chitosan solution; (C) SEM image of catalase immobilized in BN which was dispersed in water (catalase/BN) and (D) SEM image of catalase immobilized in BN which was dispersed in chitosan solution (catalase/BN/chitosan).
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3. Results and discussion 3.1. Characterization of BN, BN/chitosan, catalase/BN and catalase/ BN/chitosan In this work, BN dispersed in chitosan solution was used to adsorb catalase. Integrating the merits of BN and chitosan, BN dispersed in chitosan was more biocompatible and it offered a favorable microenvironment for the immobilization of catalase. Fig. 1A shows the sheet structure of the synthesized BN. These BN sheets aggregated together in aqueous solution with poor dispersion (inset of Fig. 1A). Fig. 1B shows that BN dispersed well in chitosan matrix. A stable milky solution was formed when 0.5% chitosan solution was used for the dispersion of the synthesized BN sheets (inset of Fig. 1B). The well-dispersed BN/chitosan can form a stable solution for a week without obvious clustering or precipitation. Chitosan is a kind of natural polyaminosaccharide. The hydrophilic/hydrophobic nature of chitosan makes it suitable to improve the solubility of BN in aqueous solution (Kisku and Swain, 2012). Its linear polyglucosamine chains have a strong tendency to adsorb on BN sheet via van der Waals forces and hydropholic interactions, and the hydrophilic amino and hydroxyl ligand helps to disperse the functionalized BN in water solution. The morphology of catalase/BN is also observed by using SEM. As shown in Fig. 1C, catalase biomolecules are excellently dispersed on BN without any aggregation, suggesting that BN is a suitable matrix for the immobilization of catalase. Fig. 1D shows that the catalase/BN/chitosan film is continuous and more uniform, which is advantageous to retain the bioactivity of proteins. UV–vis spectroscopy is a useful tool to study the possible conformational change of catalase in BN sheets. Fig. S1 shows the UV–vis absorption spectra of BN (curve a), catalase/BN (curve b) and catalase (curve c). In the case of catalase, the absorption band at about 270 nm originates from the aromatic amino acids, i.e., tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe). The changes observed in this region give evidence of the rearrangement in the globule structure of catalase (Bartoszek and Su, 2006). The position and shape of the absorption bands for catalase/BN are almost the same as those of pure catalase, suggesting that the catalase immobilized onto BN indeed maintains its native structure. 3.2. Electrochemical behavior of catalase/BN/chitosan biosensor
Fig. 2. (A) CVs of the bare GCE (curve a), BN/chitosan/GCE (curve b), catalase/ chitosan/GCE (curve c), catalase/BN/GCE and catalase/BN/chitosan/GCE (curve d) in N2-saturated phosphate buffered solution (pH 7.0, 0.1 M) at the scan rate of 100 mV/s; (B) CVs of the catalase/BN/chitosan/GCE in N2-saturated phosphate buffered solution (pH 7.0, 0.1 M) in the absence of H2O2 (a); in the presence of 100.0 μM H2O2 (b); the same as b with 5.0 μM (c) and 10.0 μM (d) of CPPU. Scan rate: 100 mV/s.
of the immobilized catalase. The catalase/BN/GCE was not stable. The modified film would deplete away from the electrode after 20 cycles. Thus chitosan was used to help the dispersion of BN in aqueous solution and to improve the stability of catalase/BN on the electrode because of its good film forming ability. It is well known that catalase-modified electrodes exhibit the electrocatalytic activities for the reduction of H2O2. Fig. 2B shows the CV response of the catalase/BN/chitosan/GCE in 0.1 M pH 7.0 phosphate buffered solution in the absence (curve a) and presence of 100.0 μM H2O2 (curve b). As compared with curve a, curve b shows larger peak current, indicating that catalase exhibits excellent bioelectrocatalytic activity for the destruction of H2O2. The general mechanism of catalase for the decomposition of H2O2 is as follows (Varma and Mattiasson, 2005):
H2 O2 + catℬFe(III) ћ H2 O+ catℬFe(IV)=O
(Reaction 1)
compound-I Fig. 2A shows cyclic voltammograms (CVs) of the bare-GCE (curve a), BN/chitosan/GCE (curve b), catalase/chitosan/GCE (curve c), catalase/BN/GCE (curve d) and catalase/BN/chitosan/GCE (curve e) in deoxygenized 0.1 M pH 7.0 phosphate buffered solution at the scan rate of 100 mV/s. For bare GCE (curve a), BN/chitosan/GCE (curve b) and catalase/chitosan/GCE (curve c), no redox peak is observed, indicating that all of them cannot undergo the redox reaction in the investigated potential range. For catalase/BN/GCE (curve d) and catalase/BN/chitosan/GCE (curve e), a pair of welldefined nearly reversible redox peaks with a formal potential of about 0.320 V versus SCE was observed. The observed formal potential for the present catalase/BN/GCE or catalase/BN/chitosan/ GCE is almost the same to the reported values for other catalasemodified electrodes (Shamsipur et al., 2012; Zhou et al., 2008). The peaks can be attributed to the redox reaction of the Cat-Fe(III)/CatFe(IV) redox couple at the active site of the immobilized catalase (Jones and Suggett, 1968; Nam, 2007; Varma and Mattiasson, 2005). This suggests that BN between the catalase and the GCE has assisted the direct electron transfer from the active site of catalase to the electrode. However, the response of catalase on BN in the absence of chitosan was smaller because of the poor dispersion of BN in aqueous solution thus caused the less amount
H2 O2 + CatℬFe(IV) = Oћ H2 O+ O2 + catℬFe(III)
(Reaction 2)
Reaction 1 is the rate-determining step. Once catalase compound-I forms (Reaction 1), it rapidly reacts with a second molecule of H2O2 to generate O2 and a water molecule (Reaction 2) thus causes the increase of the currents. Primary purpose of this study is to develop a catalase based biosensor for CPPU detection. Thus, the inhibitory effects of CPPU on the catalase catalyzed H2O2 reduction were investigated. Curves c and d in Fig. 2B show that the reduction peak current of H2O2 on catalase/BN/chitosan biosensor decreased with the increase of the concentration of the added CPPU. Thus, it is reasonable to say that the decrease in the catalytic cathodic current of the catalase/BN/chitosan biosensor in the presence of CPPU would be originated from the inactivation of the catalytic activity of catalase for H2O2 reduction. Namely, CPPU binding to catalase inhibits the reaction of H2O2 with catalase (Varma and Mattiasson, 2005), resulting in the change of the CV responses. The decrease in the peak current is directly proportional to the concentration of the inhibitor in the test solution because the percentage of inhibited enzyme that results after exposure to the
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Fig. 3. Double-reciprocal Lineweaver–Burk plot CPPU inhibition. (a) 0.0 M CPPU; (b) 2.0 μM CPPU; (c) 4.0 μM CPPU. (B) Schematic diagram of the interaction between CPPU and catalase.
inhibitor is quantitatively related to the inhibitor concentration (Amine et al., 2006). 3.3. Investigation on the type of inhibition Enzyme inhibition is a reaction between a molecule and an enzyme that blocks the action of the enzyme, either temporarily or permanently. There are several types of inhibition involving competitive, non-competitive and uncompetitive or mixed inhibition. The double-reciprocal (also known as the Lineweaver–Burk) plot was used to study the inhibition type when the concentration of H2O2 was varied at several fixed concentrations of CPPU. Fig. 3A shows a series of lines converging on the same point on the X while the Y-intercept of the plots increases with the increase of CPPU concentration. For Lineweaver–Burk plot, the slope of the resulting line is KM/Vmax, the y-intercept is 1/Vmax, and the xintercept is 1/KM. The inhibition of catalase by CPPU causes a decrease in Vmax value while KM is unaffected. The hallmarks of noncompetitive inhibition are an unchanging Michaelis constant (KM) and a decrease of the maximum velocity (Vmax) when the inhibitor is present (Fersht, 1985). So CPPU acts as a noncompetitive inhibitor by binding to catalase. Fig. 3B was used to illustrate the inhibition mechanism. In this figure, E represents the catalase enzyme; S represents H2O2 and I represents CPPU. CPPU binds to catalase at a location other than the active site. 3.4. Optimization of the variables concerning the catalase/BN/ chitosan biosensor under flow-injection conditions The experimental variables, which can affect the detection of CPPU, including the catalase to BN ratio, pH of the supporting electrolyte; applied potential and the concentration of substrate were investigated. To obtain good sensitivity of the sensor, catalase to BN ratio was optimized. Fig. S2A shows the relationship between the catalase to BN ratio and the inhibition degree of catalase by 5.0 mM CPPU. The result shows that the maximum inhibition degree was attained when the catalase to BN ratio was 8:1. It means that 1.0 mg/mL BN was the optimized concentration for the immobilization of catalase when 8.0 mg/mL catalase was used. Thus, this optimized ratio was used in all the following experiments.
Fig. S2B shows the relationship between pH and the inhibition degree of catalase by 5.0 mM CPPU. It is found the inhibition degree of catalase/BN/chitosan biosensor rises as pH increases before pH 7.0; when pH value is higher than 7.0, the inhibition degree drops as pH increases because the enzyme loses its activity. This is similar to that reported for catalase immobilized on amidoxime polyacrylonitrile nanofibrous membranes (Feng et al., 2013) and amine-functionalized graphene/gold nanoparticles composite (Huang et al., 2011). So pH 7.0 is chosen as the optimum pH of this inhibition study. The applied potential has an important influence on the sensitivity and selectivity of the biosensor. Fig. S2C shows the relationship between the applied potential and the inhibition degree of catalase by 5.0 mM CPPU. When the applied potential is decreased from 0.25 to 0.35 V, the inhibition degree increases because of the increasing driving force for the fast reduction of H2O2. When the applied potential is more negative than 0.35 V, the inhibition degree decreases with decreasing the applied potential. It can be due to a potential dependence of the activity of the immobilized catalase. In more negative potential values, catalase could be inactivated due to the formation of a high formal oxidation state of enzyme, commonly known as compound-III (Fe III) (Csöregi et al., 1993), thus caused the decrease of its catalytic effect towards H2O2. The inhibition degree also decreased. So, 0.35 V was used as the detection potential for further studies. For an inhibitor biosensor, the effect of the substrate concentration has to be adjusted carefully to obtain better results. Table S1 shows the effect of H2O2 concentration on the inhibitory influence of CPPU to catalase/BN/chitosan biosensor. The detection limit of CPPU increased with increasing the concentration of H2O2. The substrate will compete with inhibitor when the substrate concentration is high, so the increase of substrate concentration will lead to the decrease of inhibition of inhibitor on the enzyme. Otherwise, when the substrate concentration is low, the electrochemical response generated by the enzyme electrode is so weak that the determination of CPPU is hardly feasible. Therefore, we chose 1.0 mM H2O2 as the suitable substrate concentration for inhibition study. 3.5. Performance characteristics of the catalase/BN/chitosan biosensor When the constructed catalase/BN/chitosan biosensor was used at the above mentioned conditions in a flow system, the produced current would change with different concentrations of CPPU. Fig. 4 illustrates the effect of CPPU on the catalase-catalyzed H2O2 reduction peak currents. CPPU displayed increasing inhibition on the activity of catalase with increasing concentration. The inhibition curve tented to stable when the concentration of CPPU is large enough. However, the maximum inhibition value of CPPU was not 100%, which indicated that the binding sites between CPPU and enzymes could reach saturation and equilibrium. The inhibition degree (I%) of the catalase/BN/chitosan biosensor has a linear relationship with the concentration of CPPU from 0.50 to 10.0 μM. The regression equation was ΔI (μA)¼0.0083 þ 0.049C (μM) with a correlation coefficient of 0.999. The limit of the detection (LOD) for CPPU by the present catalase/BN/chitosan flow-biosensor was found to be 0.07 μM base on S/N ¼3. Table S2 listed the analytical performance of different methods for CPPU detection. This method is sensitive than the HPLC–UV method (Kobayashi et al., 2007), but is less sensitive than LC–ESI-TOFMS (Campillo et al., 2013) and ELISA methods (Suárez-Pantaleón et al., 2013). However, this value was below the approximately CPPU concentration claimed by US Code of Federal Regulations. Therefore, the proposed method is suitable for testing CPPU in real samples.
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addition method. HPLC with UV detector was chosen as a comparison method to evaluate the validity of the proposed procedure. The results obtained by the catalase/BN/chitosan biosensor and the HPLC method were summarized in Table 1. There were agreed well with each other, implying that the developed method was capable of practical applications.
4. Conclusions
Fig. 4. Flow-injection amperometric curves of catalase/BN/chitosan biosensor for different concentrations of CPPU. Inset was the calibration curves between CPPU and inhibition efficiency. The experiment was performed in 0.1 M phosphate buffered solution (pH 7.0) containing 1.0 mM H2O2 at 0.35 V; the data represented mean values and standard deviations of five measurements.
The high selectivity of the established method toward CPPU detection is important whether juice, fruit or other foods are being investigated. A systematic study of sample solutions containing a fixed amount of CPPU (1.0 mM) spiked with excess amount of some common interferences that are coexsisted with CPPU in fruits and juice samples was made to know the effect of interferences on the efficiency and selectivity of the proposed analytical method to CPPU detection. No influence on the detection of 1.0 mM CPPU was found after the addition of 1000-fold concentrations of glucose, sucrose, glycine, citric acid, Na þ and Ca2 þ . Thus the proposed method provides a possible way to monitor CPPU. The repeatability of the measurement was calculated for thirty independent runs of 1.0 μM CPPU. The relative standard deviation was 7.89%. The reproducibility of the biosensor was evaluated by the response variation of 10 biosensors, prepared on different days. The relative standard deviation was 9.73% (n ¼10), indicating good reproducibility of the sensor. The stability of the catalase/BN/ chitosan flow-biosensor was investigated by storing it at 4 °C in refrigerator and checked its response to 1.0 μM CPPU everyday. The biosensor maintained 91.3% of its initial activity after two months. Thus the presence of BN is very efficient for retaining the enzyme activity of catalase 3.6. Real sample analysis To illustrate the feasibility of the developed method for routine analysis, the method was applied to determine CPPU in different fruits and juices samples. Because the existing fruits and juices samples in the market are free of CPPU, they were fortified at different concentrations of CPPU, ranging from 1.0 to 10.0 μM. Each sample was determined in triplicate using the standard Table 1 Measurement results of CPPU in fruits and juice samples (n¼ 3). Analyte
Orange Juice Kiwifruit Watermelon Grape
Spiked (μM)
5.00 4.00 1.00 8.00
Found (mM) Catalase/BN/ chitosan biosensor
HPLC
Recovery by catalase/ BN/chitosan biosensor (%)
4.76 4.19 0.94 7.46
4.77 3.93 0.92 7.87
95.20 104.7 94.00 93.25
This work showed that the catalytic activity of catalase immobilized on BN to the reduction of H2O2 can be inhibited by CPPU. A simple and effective biosensor for the detection of CPPU has been constructed based on this inhibition effect. This biosensor has the advantages such as suitability for detecting in small volumes, fast response, low detection limit and ease of regeneration. Moreover, the presented sensor has demonstrated good sensitivity, reusability and reproducibility. These characters demonstrated the possibility of applying the biosensor, together with flow injection analysis, for the rapid determination of CPPU at low cost.
Acknowledgment The authors gratefully acknowledge the National Basic Research Program of China (2014CB931700), NSFC (21275124, 21275125 and 61222403), the Foundation of Jiangsu Educational Bureau (12KJB150022), the Qinlan Project of JiangSu Province (Grant no. 11KJB150019), and the project funded by the PAPD.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.055.
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